Cytokines comprise a variety of secreted proteins that regulate cell growth and differentiation. These factors dramatically influence the immune and inflammatory responses. Understanding the molecular basis of this regulation should provide important insights that not only shed light on disease pathogenesis but also offer new therapeutic targets. In effort to better understand cytokine signal transduction, we cloned a kinase, Jak3, mutation of which is the basis for one form of primary immunodeficiency, autosomal recessive severe combined immunodeficiency (SCID). A major objective of the laboratory continues to be to identify new patients with Jak3 mutations and to improve our understanding of the structure and function of Jak3. To this end, we have been very interested in characterizing missense mutants. For instance, we have studied in detail mutations in the pseudokinase domain, a unique feature of Jak kinases. We have demonstrated that this domain has important regulatory functions possibly by directly interacting with kinase domain. We have also been interested in delimiting the region of Jak3 that is responsible for receptor association. Through the study of one particular patient mutation we have identified a key region that is involved in receptor binding. Because of the possibility that patients who express partly functional Jak3 proteins appear to have atypical clinical presentations, we have begun analyzing patients who are catagorized as common variable immunodeficiency to see if a subset of these patients have Jak3 mutations. We have also begun investigating how Jak activation is translated to the activation of other pathways, e.g. the MAPK pathway. We demonstrated that the SH-2 containing phosphatase, SHP-2, is involved in IL-2 signaling and have identified a new IL-2 dependent tyrosine phosphorylated substrate, p98, that is associated with SHP-2, Grb2 and PI3?kinase. We have now identified this substrate to be Gab2.Our studies are also directed to understanding the function of IL-12, an essential cytokine that regulates cell-mediated immunity. IL-12 activates the transcription factor STAT4, which also has critical functions in regulating cell-mediated responses. We demonstrated that STAT4 undergoes serine phosphorylation and this is important in control of transcriptional regulation mediated by Stat4. We have performed mutational analysis of STAT4 and identified a key phosphorylation site. We have also identified candidate kinases that appear to be responsible for this event. We have also demonstrated that Stat4 is expressed in activated monocytes and have analyzed how it is activated in these cells. We have shown that Stat4 is expressed in rheumatoid synovium and have correlated its presence with the production of IFNg. To better understand the molecular basis of IL-12 action, we have also defined IL-12-inducible genes. One such gene is IRF-1 (interferon regulatory factor-1); this is notable as IRF-1 -/- mice have defective IL-12 responses. We have also identified a number of other IL-12 inducible genes using microarrays. Finally, in collaboration we have shown that the gene underlying the disease familial Mediterranean fever, MEFV is also regulated by cytokines; proinflammatory cytokines like IFNg upregulate MEFV expression whereas anti-inflammatory cytokines like IL-10, TGFb and IL-4 downregulate its expression. In a related study, we also identified the mutant gene that underlies autosomal dominant forms of periodic fever syndromes to be TNFR1, the receptor for the cytokine tumor necrosis factor. We demonstrated that patients with mutations of this gene have impaired ability to generate soluble TNFR suggesting that this is a key homeostatic mechanism. These findings have offered us new avenues to diagnose and treat these patients. - Cytokines, interleukin, interferon, signal transduction, gene regulation, kinase, immunology, immunodeficiency